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Frequency of MDR1-related p-gp overexpression in Greek Leishmania isolates

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Abstract

In this work, we investigated Greek Leishmania isolates (n = 70) for their individual MDR1-gene-related p-gp (belonging to the ABC-B subfamily of permeases) expression levels by means of flow cytometric analysis of Rhodamine 123 extrusion kinetics. Of all used isolates, 5.71 % express this drug-extruding ABC-transporter at alarming levels and are distributed widely over the country. Some 33 % of all examined isolates originated on the island of Crete though none of the strains showed vastly elevated p-gp extrusion activity, indicating a reasonable implementation of anti-leishmanial compounds in this part of the country. Compared to isolates obtained from canine tissue, human Leishmania isolates were superior both in size and in subcellular differentiation in flow cytometry. Furthermore, a specific t test confirmed verapamil hydrochloride to be a highly potent p-gp reversal agent with p < 0.0001. In a second test series, the loading of Leishmania with Rhodamine 123 was moreover reduced when occurring under influence of verapamil hydrochloride, a known p-gp reversal agent, indicating an ATP-dependant influx of the fluorescent dye and therewith the drug itself. In a final, third experiment series, it was shown that SbV does not act upon the promastigote form of Leishmania.

Comparison of a non - p-gp - expressing (upper graphs) and a p-gp – expressing (lower grpahs) Leishmania isolate: P-gp expression levels of Leishmania with reversed (grey) and non – reversed (blue) MDR 1 – related p-gp transporter activity are objectified by the extrusion of Rhodamine 123, a fluorescent dye and p-gp substrate, over the time (0’ to 120’ mins – from dark to light blue/grey)

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Acknowledgments

We wish to thank former PhD students Ippokrates Messaritakis, Panthelis Ntais and Vassiliki Christodoulou for the scientific exchange in the matter of laboratory practices. Eleni Koutala, technical assistant of the haematology department (University Hospital of Heraklion, Crete), kindly introduced us to the flow cytometer. Finally, we want to thank IMSIE (Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne) for their kind help with the mathematical interpretation of the achieved data.

Author information

Correspondence to Johannes Austrup.

Additional information

This work is a part of a doctoral medical thesis at the University of Cologne, Medical School. The experimental part was conducted at the Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine at the University of Crete (UoC, Heraklion, Greece) with the support of Ass. Prof. M. Antoniou. Former PhD student and practicing veterinarian Panthelis Ntais kindly provided the Leishmania isolates for the experiments.

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Austrup, J., Karanis, P. Frequency of MDR1-related p-gp overexpression in Greek Leishmania isolates. Parasitol Res 113, 1225–1232 (2014). https://doi.org/10.1007/s00436-014-3761-8

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Keywords

  • Visceral Leishmaniasis
  • Extrusion Activity
  • Neglect Tropical Disease
  • Verapamil Hydrochloride
  • Glucantime